AU2002243228B8 - Marine actinomycete taxon for drug and fermentation product discovery - Google Patents

Description

WO 02/47610 PCT/US01/43758 MARINE ACTINOMYCETE TAXON FOR DRUG AND FERMENTATION PRODUCT DISCOVERY FIELD OF THE INVENTION The invention relates to the discovery of a novel taxon of marine bacteria of the order Actinomycetales and the use of this taxon for the discovery and production of proteins, secondary metabolites and biomolecules for use as pharmaceutical compositions, agrichemicals, immunomodifiers, enzymes and enzyme inhibitors.

BACKGROUND OF THE INVENTION Microorganisms belonging to the class Actinobacteria, commonly called actinomycetes, reside taxonomically within the Gram-positive bacteria and are ubiquitous in terrestrial environments. Actinomycetes are a prolific source of diverse biologically active metabolites. They have been a source of a numerous useful products including pharmaceuticals, agrichemicals, low molecular weight enzyme inhibitors, immunomodifiers, and enzymes for use in a number of industrial applications, from the food industry to paper making.

These microorganisms have also been useful in agriculture as a means of pathogen protection and growth enhancement. Although many useful substances have been discovered from soil actinomycetes over the last years, the yield of novel products has drastically decreased as common soil species continually yield previously discovered metabolites. For this reason, there has been a major effort to discover new actinomycete taxa in the hope that these microorganisms will provide a new source of useful products (Bull et al., 2000).

Actinobacteria are one of a number of classes of bacteria. The class Actinobacteria can be further subdivided into six orders, including the Actinomycetales which can be broken down into 10 suborders. Classical methods for determining taxonomic novelty include morphological and WO 02/47610 PCT/US01/43758 physiological criteria such as color, presence or absence of mycelia, hyphal branch characteristics, spore pattern and motility, tolerance of variation in temperature, salinity and pH, and the ability to utilize various substrates.

Although these criteria remain an important component of taxonomic analyses, a new and more definitive method to establish strain uniqueness is 16S rDNA sequence analysis, which also provides evolutionary information on the isolate (Stackebrandt, et al., 1997).

Membership of a strain within the class Actinobacteria is indicated by 16S rDNA sequence similarity values above 80%, as determined by comparison of almost complete 16S rDNA sequences with the most deeply branching members of the class, and the presence of signature nucleotides (Stackebrandt, 1997). Signature nucleotides specific for a taxonomic group are chosen for their presence in more than 95% of the members of that group.

16S signature nucleotide sequences can be used on various taxonomic levels, from defining an order of bacteria to the subdivision of families into genera. This method provides a powerful mathematical model of bacterial evolution and an objective, rather than subjective, set of rules by which bacteria may be assigned a taxonomic status within the classification system.

Despite the fact that the oceans cover 70% of the earth's surface, all known actinomycete genera discovered to date have been land dwellers. In fact, only one marine actinomycete species has been described (Helmke and Weyland, 1984) and it belongs to a well-known terrestrial genus. Although actinomycetes have been cultured from marine sediments, it is widely believed that marine isolates are derived from dormant terrestrial spores that were washed into the sea (Goodfellow and Haynes, 1984). The "wash-in" theory was postulated because the marine isolates did not require seawater for growth, were closely related to terrestrial species, and tended to decrease in number with increasing distance from land (Goodfellow and Williams, 1983). Because many terrestrial actinomycetes can tolerate high salinity and pressure, and because of their distribution and physiology, it was concluded that most actinomycetes have been washed into the sea and collect in sediments where they can survive for long periods of time as spores (Goodfellow and Haynes, 1984). These types of studies have led to the general belief that marine actinomycetes are not significantly different from those on land and therefore of little utility as a source of novel industrial 00 products.

SUMMARY OF THE INVENTION The invention is the discovery, isolation and characterization of the first major obligate marine actinomycete taxon for which the name Salinospora gen. nov. is proposed. Members of this genus are readily recognized by a series of characteristic features including: 1. Obligate requirement of sodium (seawater) for growth.

2. Presence of at least 4 of the 5 16S rRNA signature nucleotides (Table 3) and close phylogenetic relatedness to the Salinospora clade using 16S rRNA treeing methods.

3. Morphological characteristics typically including colony color ranging from orange to brown, no or scant aerial mycelia, diffusable melanin-like pigments and spores that blacken the colony surface, hyphae that are finely branched and non-fragmenting with spores produced singly or in clusters..

4. Comparison with a deposit of a type strain at the ATCC (American Type Culture Collection 12301 Parklawn Drive; Rockville, Maryland 20852) on September 27,2000 under number ATCC PTA-2501 (Actinomycete Salinospora, strain CNH646).

The discovery of the Salinospora group refutes prior notions about actinomycetes in the marine environment and provides the first unequivocal evidence that major populations of unique, obligate marine actinomycetes occur widely in ocean sediments. Chemical studies of Salinospora group WO 02/47610 PCT/US01/43758 members have led to the isolation of novel compounds and an exceptionally high rate of biologically active extracts indicating that these microorganisms have utility for drug discovery and other industrial applications.

The invention is the use of the novel taxon for the discovery and production of proteins, secondary metabolites and other biomolecules for use in pharmaceutical compositions, agrichemicals, immunomodifiers, enzymes and enzyme inhibitors. Active molecules can be purified from the actinomycetes themselves or metabolites may be purified from the growth media. This genus is a rich source of active biomolecules with many demonstrated pharmacological activities antifungal, antimicrobial, anticancer). Extracts and products can be used in a number of assays well known to those skilled in the art to determine the activity of the various compounds derived from the actinomycetes.

The invention is the use of the genome of the taxon for the production of biomolecules in the context of the endogenous actinomycete strain or in other organisms. Genes may be expressed singly or in clusters under the control of constitutive or inducible promoters. Genes from the invention may be expressed in heterologous hosts as recombinant or over producing strains.

Other portions of the genome, such as transcriptional regulatory elements, can also be used in heterologous contexts for the control of transcription. The genome may be either wild-type or mutant. Mutations may be spontaneous or created in a random or site directed manner by methods well known to those skilled in the art.

The invention is the use of the taxon for the production of gene products from heterologous organisms. Genes may be inserted either singly or in clusters into the actinomycete strains of the invention for expression of proteins, secondary metabolties or other biomolecules. Compounds may be isolated from the actinomycetes or the growth media.

The invention is the use of the taxon for pathogen and pest protection, insecticides, herbicides, microbiocides, growth promotion in agriculture and -4- WO 02/47610 PCT/US01/43758 aquaculture applications. Actinomycetes of the instant invention can compete with harmful micro-organisms in the environment of the plants providing a non-toxic means of protecting plants.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be better understood from the following detailed description of an exemplary embodiment of the invention, taken in conjunction with the accompanying drawings in which like reference numerals refer to like parts and in which: Figure 1. Structure of salinosporamide A, the first novel, bioactive metabolite obtained from the Salinospora group. The isolation of this compound proves that the Salinospora group is a resource for unique, biologically active metabolites. The producing strain was cultured in a seawater-based medium and the compound was obtained in pure form following a series of chromatographic steps. The structure of salinosporamide A was elucidated using 1D and 2D nuclear magnetic resonance and high resolution mass spectral data analyses.

Figure 2. Phylogenetic dendogram created using the neighbor joining method showing seven diverse members of the Salinospora clade along with representatives from all genera officially belonging to the Micromonosporaceae family (Koch, et al; 1996). The distance bar indicates 1 nucleotide substitution per 100 nucleotides. The Salinospora clade is shown in the bracket. In parenthesis along side of the Salinospora strain designations are the locations and dates of the expeditions from which the isolates were obtained. The Salinospora group to date includes 400-500 isolates that showed the characteristic features 1 and 3 (listed in the Background section). These strains are from three expeditions that were grouped first by location and date obtained, then by morphological diversity within each group. From each of these sub-groups, isolates were chosen for almost complete 16S rRNA gene sequencing (greater than 95% of the gene).

WO 02/47610 PCT/US01/43758 Phylogenetic programs contained in the Phylip and Clustal W packages were used for analysis and the tree was drawn using Treeview 1.6.1.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS Salinospora strains can be consistently isolated from marine sediments and are distinguished by characteristic signature nucleotides, an obligate requirement of seawater more specifically, Na') for growth, and morphological characteristics. Isolates belonging to this group were obtained on five separate occasions from tropical to subtropical, near-shore sediments collected from the Atlantic Ocean, the Red Sea and the Sea of Cortez indicating a world-wide distribution. In these studies, a total of 147 independent sediment samples were evaluated and 51 of these yielded a total of 182 Salinospora isolates of which seven were subjected to in-depth physiological and phylogenetic evaluation (Table Subsequently, over 1000 strains have been obtained from additional collections.

Natural location of strains. Select, diverse isolates representing over 1000 strains with Salinospora morphology were used for in-depth phylogenetic and physiological analyses. Nearly complete 16S rDNA sequences of the entire gene) were obtained for all strains listed. Note that CNB394 and CNB512 are marine-derived Micromonospora isolates and were carried through analyses to illustrate fundamental differences between Salinospora and Micromonospora genera.

Table 1.

Strain Year and Habitat descrip. Genus location and depth CNH643 1999 Bahamas, Coarse sand, Salinospora Sweetings Cay 1m CNH646 1999 Bahamas, Spur and grove, Salinospora Andros Island 10 m WO 02/47610 PCT/US01/43758 CNH725 2000 Red Sea, Coarse sand, Salinospora Sha'b el utal 20 m CNH898 2000 Bahamas, Coarse sand, Salinospora Little San 30 m Salvador CNH964 2000 Sea of Coarse sand, Salinospora Cortez 30 m Caleta Partida CNB440 1989 Bahamas, Spur and grove, Salinospora Chub Cay 20 m CNB536 1989 Bahamas, Coarse sand and Salinospora Acklins Island seagrass, CNB394 1989 Bahamas, Coarse sand and Micromonospora Chub Cay seagrass, 1m CNB512 1989 Bahamas, Spur and grove, Micromonospora San Salvador Is. All of the 182 Salinospora strains tested failed to grow on an agar medium when seawater was replaced with deionized water. Seven phylogenetically diverse strains were further characterized and shown to require sodium for growth (Table a physiological characteristic commonly associated with obligate marine bacteria. Sodium requirements have been studied extensively in Gram-negative marine bacteria and are indicative of highly evolved marine adaptations such as a respiration-dependant sodium ion pump and/or a sodium dependent membrane transport mechanism. The requirement of seawater (sodium) for growth is extremely rare in Grampositive bacteria and has never before been reported for an actinomycete with the exception of Rhodococcus marinonascens (Helmke and Weyland, 1984) Assay for sodium dependent growth. Physiological growth analysis illustrating fundamental growth differences between Salinospora (in bold) and marine-derived Micromonospora isolates (CNB394, CNB512) are shown in WO 02/47610 PCT/US01/43758 Table 2. Tests were performed on medium M1 which was found to be optimal for the growth and maintenance of Salinospora and Micromonospora genera.

Isolates were screened using a sterile cotton swab to inoculate macerated, vegetative mycelia onto each analytical medium which was then incubated at 25-280C for six to eight weeks. Growth was checked periodically using a Leica stereoscope at 10-64X magnification. All strains grew equally well in natural seawater (NSW) or artificial seawater (ASW No detectable growth was observed for any of the Salinospora isolates on M1 prepared with de-ionized water The two Micromonospora isolates, CNB394 and CNB512, grew better on M1 DI water than on the seawater-based medium.

Sodium growth requirements were tested on M1 prepared with ASW in which all sodium sources were replaced with equimolar amounts of potassium (M1, ASW The sodium concentration in seawater of salinity 35 (used for M1, NSW medium in this study) is 450 mM. In order to determine the upper limits of sodium chloride tolerance, strains were tested for growth on M1 DI water in which sodium chloride was added to yield final sodium concentrations of 600 and 1000 mM. The Salinospora isolates showed no growth at these elevated sodium levels whereas growth was clearly evident for the marine-derived Micromonospora CNB394 and CNB512.

Table 2.

Medium Isolates Tested CNB CNB CNB CNB CNB CNH CNH CNH CNH 394 512 440 536 643 646 721 898 964 M1,

NSW

M1,

DI

H

2 0 WO 02/47610 PCT/US01/43758 M1

ASW

Na M1

ASW

K+

600 mM [Na'] 1000 mM [Na'] Salinospora isolates are proving to be a remarkable source of biologically active secondary metabolites. Thus far, of the 105 strains examined, 86% yielded culture extracts with significant cancer cell cytotoxicity (ICo 5 values ranging from 0.004 16.4 micrograms/ml against the HCT-116 human colon carcinoma cell line). Significant antifungal and antibiotic activities have also been observed from the extracts of cultures grown under various conditions with 30% yielding MIC values of 19.5 micrograms/ml or less against amphotericin resistant Candida albicans and 35% yielding extract minimum inhibitory concentration (MIC) values of 25 micrograms/mi or less against vancomycin resistant Enterococcus faecium.

Thin layer chromatography and liquid chromatography/mass spectrometric analyses, as well as Repetitive Extragenic Palindrome Polymerase Chain Reaction (REP-PCR), indicate considerable strain to strain chemical and genetic diversity. Bioassay-guided fractionation of one active extract has led to the isolation of a novel series of metabolites that includes a potent cytotoxin (ICs, 10 ng/ml against the HCT-116 human colon carcinoma cell line) that has been named salinosporamide A (Figure This molecule is most closely related to clasto-lactacystin beta-lactone (also called k omuralide), the intermediary hydrolysis product of lactacystin, an anti- J microbial product. Salinosporamide A represents the first natural product to Sbe discovered that possesses a fused beta-lactone gamma-lactam bicyclic ring and is a highly potent anticancer agent.

The Salinospora group was initially recognized after phylogenetic 00 Ccharacterization of sediment-derived actinomycetes isolated during an tc expedition to the Bahamas. Partial 16S rDNA gene sequences from eight Smorphologically diverse strains indicated the presence of four signature nucleotides between positions 198 to 1424 of SEQ ID Nos:3, 4 and 5, or Snucleotide positions 207-468 (based on the E. coli 16S rRNA numbering system which is well known to those skilled in the art; see also Table 3 which utilizes the E coli 16S rRNA numbering system in comparing the Salinospora actinomycetes of the present invention to those of other Micromonosporaceae genera). These signatures nucleotides have subsequently been found in all partially sequenced Salinospora strains. Two strains showing the highest phylogenetic diversity (CNH643 and CNH646) were sequenced nearly in their entirety (GenBank accession numbers AY040619 (SEQ ID NO:3) and AY040620 (SEQ ID NO:4), respectively) and found to possess one additional signature nucleotide (position 1456) that is also characteristic of this group (Table Phylogenetic analyses of aligned sequences from these strains indicate that they form a distinct and coherent clade within the Micromonosporaceae (Figure Signature nucleotides unify this clade and a high bootstrap value supports clear separation from the nine currently described genera within the family.

16S rRNA signature nucleotides. 16S rRNA signature nucleotides for the genus Salinospora and all nine currently accepted genera within the Micromonosporaceae are shown in Table 3. Forty-five diverse Salinospora isolates were partially sequenced and confirmed to have all four signature nucleotides at positions 207-468. The signature nucleotide at position 1456 was discovered after subsequent 3'sequencing of the 16S rRNA gene from several (20) Salinospora isolates. These are original signatures observed in this study (in addition to those previously published by Koch et al. 1996) that define the coherence of the Salinospora clade and separate it from other Smembers of the family. Signature nucleotides were aligned to E. coli positions 27-1492 using all existing members of the Micromonosporaceae in the 00

(N

Ribosomal Database Project. Members of the genus Salinospora show closest homology to Micrononospora olivasterospora (97.1-97.7% similarity), Ithe most deeply rooted member of that genus, with whom they share eight of 12 previously published signature nucleotide positions. Thus Salinospora oo strains are more highly diverged from their closest phylogenetic neighbor than the recently described genus Verrucosispora gifornensis which shows 98.0% similarity to Micromonospora olivasterospora and shares 11 of 12 previously published signature nucleotides.

Table 3 Relative All Other Nucleotide Nucleotide Micormonosporaceae Salinospora Positions of Position of E.Coli genera SEQ ID NOs: 16S RNA 4 and 207 A 198 366 C 351 467 U 442 468 A U 443 1456 A G 1423 A follow-up study was undertaken in the Bahamas to determine the persistence of the Salinospora group. From 20 samples collected from four transects (0-30 355 actinomycetes were observed and over 90% of these displayed characteristic Salinospora morphologies suggesting that this group may be the numerically dominant actinomycete in marine sediments. Of those observed, 100 strains were isolated for further study. The average numbers of Salinospora colony-forming units (cfu's) ranged from 1.2-2.3 x 10 3 cfu's/ml sediment. Over 50% of the Salinospora isolates appeared on a low nutrient medium (M4) indicating the importance of using appropriate isolation techniques. Thirteen representatives of eight different colony morphotypes -11 were partially sequenced and the most phylogenetically diverse isolate (CNH898) was sequenced nearly in its entirety (GenBank Accession number AY040622) (SEQ ID An examination of 30 actinomycetes with Salinospora morphological characteristics that were isolated from the Bahamas in 1989 (Jensen et al, 00 eC 1991) revealed that all but two of these strains had an obligate requirement of seawater for growth. Ten seawater requiring strains representing six different morphotypes were partially sequenced and found to possess the five Salinospora signature nucleotides between positions 207-468 (Table The nearly complete 16S rDNA sequence of two of these (CNB440 and CNB536, Gen Bank Accession numbers AY040617 (SEQ ID NO:6) and AY040618 (SEQ ID NO:7), respectively) indicates that they are diverse members of the Salinospora clade (Figure Thus, strains belonging to this new taxon have been isolated from near-shore Bahamian sediments on three separate occasions over an 11-year period indicating that they are persistent members of the sediment bacterial community.

The two strains that did not require seawater for growth (CNB394 and CNB512) but had colony morphologies similar to Salinospora were found to lack the Salinospora signatures in Table 3. Analyses of the almost complete 16S rDNA sequence of these strains showed 99.6-99.9% similarity to Micromonospora aurantiaca str. W2b and the presence of all of the signature nucleotides previously published for the genus Micromonospora (Koch et al, 1996). The phylogenetic dendogram clearly shows that CNB394 and CNB512 are members of the genus Micromonospora (Figure Micromonospora isolates have been reported from marine sediments (Takizawa et al, 1993), including deep-sea samples (Weyland, 1981), however, unlike Salinospora, this genus is well known from terrestrial soils and seawater-requiring strains have not been reported.

From extended supra-littoral transects (10 locations, 30 samples) made in the Bahamas (2000 expedition), over 1000 actinomycete colonies were -12- C observed including low numbers of Micromonosporaceae (ca. however Snone of these required seawater for growth. The inability to recover SSalinospora strains from supra-littoral samples supports the observation that these bacteria are restricted to the marine environment.

To determine if Salinospora members had a broader distribution, 00 sediments were collected from the Red Sea and the Sea of Cortez. From 42 Red Sea sediment samples, 22 isolates with Salinospora morphologies and an obligate requirement of seawater for growth were obtained. Six isolates representing 4 major morphotypes were partially sequenced and the almost complete 16S rDNA sequence of one strain (CNH725, GenBank Accession number AY040621) (SEQ ID NO:8)_is represented in Figure 2. From 36 sediments collected in the Sea of Cortez, 20 seawater-requiring actinomycete strains were isolated and all of these possessed Salinospora morphological characteristics. Eight strains representing five different morphotypes were partially sequenced and the phylogenetically diverse isolate CNH964 (GenBank Accession number AY040623) was sequenced almost in its entirety. These results clearly indicate that Salinospora members are widely distributed in marine sediments.

Phylogenetic analyses and physiological characteristics indicate that the Salinospora clade represents a new genus within the family Micromonosporaceae. Although it is unlikely that the diversity within this genus has been revealed in the present study, intra-group 16S rDNA sequence similarity and a robust clade topology indicate that this genus is comprised of multiple species (Figure Placement of the genus Salinospora within the family Micromonosporaceae is supported by the presence of a complete set of family-specific 16S rDNA signature nucleotides (Stackelbrandt, 1997).

Despite evidence that actinomycetes can be recovered from deepocean sediments, only one marine species has been described (Helmke and Weyland, 1984) and the inclusion of this group within the autochthonous -13- WO 02/47610 PCT/US01/43758 marine microbiota has not been widely accepted (Bull et al., 2000). Our data provide the first conclusive evidence for the widespread and persistent occurrence in marine sediments of unique populations of obligate marine actinomycetes. Phylogenetic and physiological evidence indicate that these actinomycetes comprise a new taxon and the generic epithet Salinospora gen.

nov. has been proposed. Salinospora strains are a prolific source of biologically active secondary metabolites that are useful for a variety of applications.

EXAMPLE 1 Sample collection and bacterial isolation. Samples of the top 1 cm of sediment were collected by SCUBA and processed by either stamping, dilution and heat-shock or both methods. Dilution and heat-shock was carried out as follows: 1 ml of wet sediment was added to 4 ml of sterile seawater, heated for six minutes at 55 0 C, shaken vigorously, and dilutions of 10 2 to 10 4 were inoculated onto agar media (M1-M4). For stamping, 10 ml of wet sediment were aseptically placed into a sterile aluminum dish, dried (ca. 24 hours) in a laminar flow hood, ground lightly with a pestle, pressed into a sterile foam plug (14 mm in diameter) and inoculated onto agar media (M1- M4) by stamping 8-9 times in a clockwise fashion giving a serial dilution effect.

All isolation media were prepared with 100% filtered natural seawater.

Actinomycetes generally appeared after 4-6 weeks of incubation at 25-28 0

C

and were considered as any colony with a tough leathery texture, dry or folded appearance and branching filaments with or without aerial mycelia. All isolation media had final concentrations of 100 micrograms/ml cycloheximide and 5 micrograms/ml rifampicin added after autoclaving.

Media were prepared by methods well known to those skilled in the art and all contain seawater. Recipes for media are as follows: Ml: 10 grams starch, 4 grams yeast extract, 2 grams peptone, 18 grams agar, 1 liter natural seawater; M2: 6 ml glycerol, 1 gram arginine, 1 gram K 2

HPO

4 0.5 grams -14- WO 02/47610 PCT/US01/43758 MgSO, 18 grams agar, 1 liter natural seawater; M3: 6 grams glucose, 2 grams solubilized chitin, 18 grams agar, 1 liter natural seawater; M4: 2 grams solubilized chitin, 18 grams agar, 1 liter natural seawater; M5: 18 grams agar, 1 liter natural seawater.

EXAMPLE 2 DNA purification, amplification, sequencing and phylogenetic analyses.

Genomic DNA was prepared as follows: 10 mg of vegetative mycelia grown on M1 agar for 2-4 weeks at 25-28 0 C was macerated and an aqueous cleared lysate, created by standard methods, was precipitated with 0.7 volumes of isopropanol. The resultant DNA pellet was then washed with 70% ethanol and resuspended in 10 mM Tris buffer (pH 8.5) to a final concentration of 100 ng/ml. 16S rDNA sequencing templates were amplified from 10-50 ng of genomic DNA template by the PCR using the primers FC27 AGAGTTTGATCCTGGCTCAG) (SEQ ID 1) and RC1492 TACGGCTACCTTGTTACGACTT) (SEQ ID PCR products were purified with a Qiagen QIAquick PCR clean-up kit using the manufacture's protocols.

Partial sequences of morphologically diverse strains were obtained from nucleotides 80-480 colinumbering system) using the FC27 primer. Select 16S rDNA amplicons were sequenced almost in their entirety on both top and bottom strands using a total of ten primers. The ten contigs were then assembled yielding gene sequences of 1479 to 1483 unambiguous nucleotides. Hypervariable regions in the 16S rDNA sequences were excluded yielding a total of 1408 aligned nucleotides. 16S rDNA similarity values were calculated by the RDP similarity matrix online analysis and compared to the three nearest neighbors in the RDP database. Sequences were aligned to the secondary structure of members of the Micromonosporaceae in the RDP (Maidak et al, 2001) using the BioEdit software (Hall, 1999). Phylogenetic analyses were performed using the neighbor-joining and parsimony based algorithms in the Clustal W software WO 02/47610 PCT/US01/43758 and PHYLIP software packages, respectively (Thompson et al., 1994; Felsenstein, 1993). The dendogram (Figure 2) was drawn using Treeview 1.6.1 (Page, 1996).

EXAMPLE 3 Genetic-analysis by Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR). The genetic diversity of Salinospora strains was analyzed using REP-PCR (Versalovic et al., 1991). This technique, when applied to the Salinospora group, involves the use of total genomic DNA as a template and PCR primers specific for repetitive sequences present in the genomes of high G+C content Gram-positive bacteria. The length of the PCR products for any one strain will vary with the position of the repetitive sequences in the genome and result in a population of amplicons of various lengths that when separated on an agarose gel create strain-specific banding patterns. This high throughput method allows for the detection of genetically distinct strains and is more sensitive than 16S rRNA gene analyses as a method to assess genetic diversity. REP-PCR banding patterns are used to sort strains into distinct groups that can produce different gene products.

Grouping of strains based on REP-PCR banding patterns correlate well with groups based on the production of secondary metabolites.

EXAMPLE 4 Production. and isolation of useful products. Salinospora strains were cultured in multiple sea-water based media including M1 and CKA (starch 5 g, fish hydro-solubles 4 ml, menhaden meal 2 g, kelp powder 2 g, chitosan 2 g, seawater 1 An adsorbent resin (XAD-16) was added to the fermentation 24 hours prior to harvest (day 11). The resin was collected by filtration, rinsed with deionized water, and eluted with acetone. Alternatively, cells were collected by filtration, freeze dried and extracted with acetone. The extract was concentrated by rotary evaporation and the residue subjected to C-18 WO 02/47610 PCT/US01/43758 flash chromatography followed by HPLC. The structures of novel fermentation products were resolved using a variety of methods including oneand two-dimensional NMR and mass spectroscopy.

EXAMPLE Antibacterial assay. Extracts from cultured Salinospora strains were tested using standard methods to demonstrate their antibiotic activity against Grampositive and Gram-negative bacteria. The method used to test against Staphylococcus aureus is detailed below. Similar methods are used to test for antimicrobial activity against other organisms. Extracts were compared to known antibiotics and relative activity levels determined. Extracts with potent antibiotic activity were further analyzed for the presence of novel metabolites.

Briefly, cultures of S. aureus were grown overnight to stationary phase.

The number of bacteria per ml was calculated and a uniform number of bacteria were plated into individual wells containing fresh media. Compounds of interest, including known antibiotic agents Oxacillin in DMSO at 0.04 mg/mL), were added to a single row of wells and serially diluted down the plate to determine the concentration required to kill the bacteria. Plates were incubated overnight at 37 0 C to allow for cell growth. Samples were read in an automated plate reader at 600 nm and MIC concentrations were determined.

EXAMPLE 6 Antifungal assay. Extracts from cultured Salinospora strains were tested using standard methods to demonstrate antifungal activity against Candida albicans. Extracts were compared to known antibiotics and their relative activities determined. Extracts with potent antifungal activity were further analyzed for the presence of novel metabolites.

Briefly, a culture of C. albicans was grown overnight to stationary phase. The number of cells per ml was calculated and the suspension was diluted and added to individual wells of 96-well plates. Alamar blue was -17- WO 02/47610 PCT/US01/43758 added to each well as an indicator of viability. Test extracts were added to a single row of wells and serially diluted down the plate to determine the concentration required to kill the fungal cells. Known antifungal agents such as amphotericin were used as a control. Plates were incubated for 12-15 hours at 370C. Cell concentrations were determined using an automated plate reader at 600 nm and MIC concentrations were determined.

EXAMPLE 7 Assay for the inhibition of growth of colon carcinoma cells and of ovarian cancer cells in vitro. The cytotoxicity of extracts from cells or culture media were assessed in vitro against the human colon carcinoma cell line HCT116 and the human ovarian carcinoma cell line A2780 by MTS assay. Cells were plated at 4,000 cells per well in 96 well microliter plates and, after 24 hours, the extract (dissolved in DMSO or other appropriate solvent) was added and serially diluted. The cells were incubated with the compound at 370C for 72 hours, then the tetrazolium dye MTS was added to a final concentration of 333 pg/ml and the electron coupling agent phenazine methosulfate was added to a final concentration of 25 pM. Once reduced, MTS is converted into a water insoluble blue crystal formazan and that was read at an absorbance at 490 nm with a microplate reader. As dead cells are unable to reduce MTS, the amount of formazan is correlated to the number of viable cells. The IC0o, the drug concentration required to inhibit proliferation of 50% of the cells, was used as a measure of efficacy.

EXAMPLE 8 Anti-Herpes Simplex Virus (HSV-1) assay. Antiviral activity can also be determined using an MTS assay. Vero cells were plated into duplicate 96-well plates for infection with virus and cytotoxicity control. One plate of cells was incubated with virus for an hour at 370C. Both plates were overlaid with a series of concentrations of the extract of interest and plates were incubated -18- WO 02/47610 PCT/US01/43758 for five days. MTS solution was added to the plates and the plates were incubated for three hours as described above. Absorbance at 490 nm was read with a microplate reader and correlated to antiviral activity and cellular toxicity.

EXAMPLE 9 Chemical mutagenesis of Salinospora strains to generate overproducing strains. Chemical mutagenesis of Salinospora strains can be performed to generate strains that overproduce a desired product. For example, a strain that produces an antibiotic at a low level is treated with ethylmethylsulfonate (EMS) during the mid-log growth phase. Mutagenized cultures are streaked onto plates to allow for the isolation of individual clones. From the individual clones, cultures are grown and the antibiotic, in a crude or pure form, is isolated. The relative yields of the compounds of interest produced by the mutagenized strains are compared to the original strain to select an overproducing strain.

EXAMPLE Heterologous gene expression. Actinomycete strains have been useful as hosts for the production of secondary metabolites from other more slowly growing organisms (Tang, et al., 2000). Genes, either singly or in clusters, can be expressed in Salinospora strains for the production of proteins or secondary metabolites. Methods for transferring nucleic acids into bacteria are well known by those skilled in the art.

EXAMPLE 11 Gene cluster isolation and expression. The synthesis of a number of actinmycete antibiotics actinorhodin, frenolicin, granaticin, griseusin, octatetracycline, and tetracenomycin) are produced by clustered polyketide synthetase (PKS) genes (Hopwood, 1995). PKS genes are classified into two WO 02/47610 PCT/US01/43758 types of large mutifunctional proteins. In PKS type I genes, the substrate progresses through a number of active sites on a single protein. In PKS type II genes, multiprotein complexes are produced and the substrate progresses from one protein to the next. PKS type II genes have been cloned and expressed in heterologous systems, either in their native groupings or in novel combinations. Combining genes for the synthesis of secondary metabolites from Salinospora with genes from other actinomycetes provides a novel method of biologically assisted combinatorial chemistry that can lead to the production of novel small molecules. Also, Salinospora biosynthetic genes can be shuffled and expressed in an heterologous host leading to the production of new metabolites. PKS genes are not the only ones that occur in modules. For example, non-ribosomal peptide synthetases are modular as well, and are frequently present in the actinomycetes. Biosynthetic gene clusters from the novel Salinospora group can be used as genetic feedstock for the expression of novel molecules in heterologous strains or for the overproduction of native and recombinant gene products.

EXAMPLE 12 Assay for anti-inflammatory activity. Extracts from Salinospora cultures are tested by measuring inhibition of phorbol-induced inflammation (edema) in a mouse ear assays. This is a conventional test which has been accepted as demonstrating a compound's effectiveness in reducing inflammation. The compound is topically applied in acetone to the inside pinnae of the ears of mice in a solution containing an edema-causing irritant, i.e. phorbol 12myristate 13-acetate (PMA). PMA alone (2 microgram per ear) or in combination with varying amounts of the extract is applied to the left ear mice per treatment group) while an acetone (control) is applied to the right.

After a 3-hour and 20-minute incubation at 23 0 C, the mice are sacrificed, the ears removed, and bores taken and weighed. Edema is measured by subtracting the weight of the right ear (control) from the weight of the left ear WO 02/47610 PCT/US01/43758 (treatment). The results are recorded as a percent decrease (inhibition) or percent increase (potentiation) in edema relative to PMA.

EXAMPLE 13 Enzyme inhibition assay. Extracts from Salinospora strains could be tested for their ability to inhibit enzyme activity. Extracts could be prepared as described above and serial dilutions of the extract added to enzyme-substrate mixtures to determine an IC,, for the reaction.

EXAMPLE 14 Enzyme activity assay. Assays for enzyme activity can be tested by growing Salinospora strains in the presence of substrates of interest including, but not limited to chitin, lignin, cellulose, and other recalcitrant biopolymers, etc.

Depending on the substrate, assays can be performed to determine the amount of substrate remaining or the amount of product produced.

EXAMPLE Agriculture/aquaculture protection assay. Assays for the protection of plants from pathogens and general growth enhancement can be performed in a standard greenhouse trial. The strain of interest can be applied to the plant directly or incorporated into the growth media. Plants could be challenged by subjecting them to a pathogen and comparing their growth to control groups treated with a pathogen alone, treated with a Salinospora strain alone, or untreated. Rates of growth could be compared to select for strains with the desired activities.

Although an exemplary embodiment of the invention has been described above by way of example only, it will be understood by those skilled in the field that modifications may be made to the disclosed embodiment WO 02/47610 PCT/US01/43758 without departing from the scope of the invention, which is defined by the appended claims.

REFERENCES

Bull, A. A. C. Ward, and M. Goodfellow. 2000. Search and discovery strategies for biotechnology: The paradigm shift. Microbiology and Molecular Biology Reviews. 64:573-606.

Felsenstein, J. PHYLIP (Phylogeny Inference Package) version (Distributed by the author. Department of Genetics, University of Washington, Seattle., 1993).

Goodfellow, and J. A. Haynes. 1984. Actinomycetes in marine sediments, p. 453-472. In L. Ortiz-Ortiz, L. F. Bojalil, and V. Yakoleff Biological, biochemical, and biomedical aspects of actinomycetes.

Academic Press, Inc. Orlando.

Goodfellow M. and S.T. Williams. 1983. Ecology of actinomycetes. Ann. Rev.

Microbiol. 37:189-216.

Helmke, E. and Weyland, H. 1984. Rhodococcus marinonascens sp. nov., an actinomycete from the Sea. Int. J. Syst. Bacteriol. 34:127-38.

Hopwood, D.A. 1995. Genetic manipulation of Streptomyces polyketide synthase genes for novel secondary metabolite production. FEMS Microbiol. Rev. 16:233-4.

-22- WO 02/47610 PCT/US01/43758 Jensen, Dwight, and Fenical, W. 1991. Distribution of actinomycetes in near-shore tropical marine sediments. Appl. Environ. Microbiol 57:1102-8.

Koch, R. M. Kroppenstedt, F. A. Rainey, and E. Stackebrandt. 1996. 16S ribosomal DNA analysis of the genera Micromonospora, Actinoplanes, Catellatospora, Catenuloplanes, Couchioplanes, Dactylosporangium, and Pilimelia and emendation of the family Micromonosporaceae.

International Journal of Systematic Bacteriology. 46:765-768.

Page, R. D. M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357-358.

Stackebrandt, F. A. Rainey, and N. L. Ward-Rainey. 1997. Proposal for a new hierarchic classification system, Actinobacteria classis nov.

International Journal of Systematic Bacteriology. 47:479-491.

Takizawa, Colwell, R. R. Hill, R. T. 1993. Isolation and diversity of actinomycetes in the Chesapeake Bay. Applied and Environmental Microbiology 59, 997-1002.

Tang, Shah, Chung, Carney, Katz, Khosla, and Julien, B.

2000. Cloning and heterologous expression of the epothilong gene cluster. Science. 287:640-2.

Thompson, J. Higgins, D. G. Gibson, T. J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment -23- WO 02/47610 PCT/USO1/43758 through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673-4680.

Versalovic, T. Koeuth, and J. R. Lupski. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823-6831.

Weyland, H. 1981. Distribution of actinomycetes on the sea floor.

Actinomycetes, Zbl. Bakt. Suppl. 11:185-193.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

-24-

Claims (21)

1. An isolated marine actinomycete having an obligate requirement of sodium for growth, wherein the marine actinomycete is a strain of Salinospora Scomprising 16SrRNA sequences as set forth in SEQ ID NO:4 and SEQ ID 00 c 2. An isolated marine actinomycete strain of Salinospora of ATCC deposit 0 No. PTA-2501 or a variant or mutant thereof.

3. An isolated marine actinomycete of claim 1, wherein the strain of c- Salinospora is obtained from sediment.

4. A method for producing a biomolecule comprising: growth of a strain of a marine actinomycete with an obligate requirement of sodium for growth according to any one of claims 1-3, wherein the biomolecule is produced by the strain of Salinospora; collecting the strain of Salinospora or the sodium containing growth media containing the biomolecule; and extracting the biomolecule from the strain of Salinospora or the medium containing growth media, thereby producing the biomolecule. The method of claim 4, wherein the biomolecule is selected from the group consisting of a pharmaceutical agent, an antibiotic agent, and antifungal agent and an anti-cancer agent.

6. The method of claim 5, wherein the biomolecule is an anti-cancer agent.

7. The method of claim 5, wherein the biomolecule is an antibiotic agent.

8. The method of claim 5, wherein the biomolecule is an antifungal agent.

9. The method of claim 4, wherein the biomolecule is a product of a gene from a heterologous organism. A method for drug discovery comprising: growth of a strain of a marine actinomycete with an obligate oo c requirement of sodium for growth according to any one of claims 1-3 cand collecting the strain of Salinospora or the sodium containing growth c media containing the biomolecule; S(b) extracting the biomolecule from the strain of Salinospora or the medium containing growth media; and analyzing the biomolecule or sodium containing growth media for pharmacological activity.

11. The method according to claim 10, wherein analyzing the biomolecule comprises an assay for antibacterial activity.

12. The method according to claim 10, wherein analyzing the biomolecule comprises an assay for anti-viral activity.

13. The method according to claim 10, wherein analyzing the biomolecule comprises an assay for anti-cancer activity.

14. The method according to claim 10, wherein analyzing the biomolecule comprises an assay for anti-fungal activity. The method according to any one of claims 4-14, wherein when the growth media is collected, the sodium concentration of the growth medium is 450 nM. -26- N 16. A salinosporamide A compound isolatable from a strain of Salinospora, wherein the strain is marine actinomycete with an obligate requirement of Ssodium for growth according to any one of claims 1-3.

17. The salinosporamide A compound according to claim 16, wherein the cN Salinospora comprises 16SrRNA sequences as set forth in SEQ ID NO:4 and 0 SEQ ID NO:5, and wherein SEQ ID NO:4 and SEQ ID NO:5 have signature Snucleotides not found in other Micromonosporacea rRNAs. O C 18. The salinosporamide A compound of claims 16 or 17, wherein the strain of Salinospora is CNH646.

19. The salinosporamide A compound according to any of claims 16-18, wherein signature nucleotides are numbered and identified in accordance to the E. coli 16S rRNA sequence, and wherein a uridine or cytosine at position 207 is an adenosine; an adenosine or guanine at position 366 is a cytosine; an adenosine or guanine at position 467 is a uracil; an adenosine at position 468 is a uracil; and adenosine at position 1456 is a guanine. A 4-chloroethyl-5-methyl- 1 -((cyclohex-2-enyl)(hydroxy)methyl)-6-oxa- 2- azabicyclo(3.2.0)heptane.-3,7-dione isolated from a marine actinomycete with an obligate requirement of sodium for growth according to any one of claims 1-3.

21. A 4-chloroethyl-5 -methyl- 1 -((cyclohex-2-enyl)(hydroxy)methyl)-6-oxa- 2- azabicyclo(3.2.0)heptane-3,7-dione according to claim 20, wherein the marine actinomycete is a strain of Salinospora comprising 16SrRNA sequences as set forth in SEQ ID NO:4 and SEQ ID

22. A 4-chloroethyl-5-methyl- 1 -((cyclohex-2-enyl)(hydroxy)methyl)-6-oxa- 2- azabicyclo(3.2.0)heptane-3,7-dione according to claim20, wherein SEQ ID NO:4 and 5 have signature nucleotides not found in other Micromonosporacea rRNAs, and wherein a uridine or cytosine at position 207 is an adenosine; an adenosine or guanine at position 366 is a cytosine; an -27- adenosine or guanine at position 467 is a uracil; an adenosine at position 468 O Sis a uracil; and adenosine at position 1456 is a guanine.

23. The 4-chloroethyl-5-methyl-1 -((cyclohex-2.-enyl)(hydroxy)methyl)-6- c oxa-2- azabicyclo(3.2.0)heptane-3,7-dione of claim, wherein the strain of Salinospora is CNH646. 0o c 24. A pharmaceutical composition comprising an effective amount of a Spharmaceutically acceptable carrier and a compound of any one of claimsl 6- 23. c 25. Use of a strain as claimed in any one of claims 1-3 or a compound as claimed in any one of claims 16-23 in the preparation of a medicament for the treatment of one or more of cancer, bacterial infection and fungal infection.

26. An isolated actinomycete strain substantially as herein described with reference to the examples.

27. A method for producing a biomolecule, the method substantially as herein described with reference to the examples.

28. A compound or pharmaceutical composition of any one of claims 16-23 substantially as herein described with reference to the examples or drawings.

29. The method of any one of claims 4-9, wherein the biomolecule is Salinosporamide A. A Salinosporamide A compound produced by the method of any one of claims 4-9.

31. A Salinosporamide A compound isolated from a strain of Salinosora, wherein the strain is a marine actinomycete with an obligate requirement of sodium for growth according to any one of claims 1-3. -28-